SU1586520A3 - Method of producing n-nitroso-n-(beta-chloroethylcarbamoylpeptides)) or their salts - Google Patents

Abstract

Compounds of the general Formula I [Cl-CH2-CH2-@@CO-NH]nR (I) wherein R is a group derived from any oligopeptide by removing a terminal amino group, or - if the oligopeptide comprises a diamino carboxylic acid - the amino group in the omega -position, or the terminal and omega -amino groups; and n stands for the integral number 1 or 2 and acid addition salts thereof and a process for the preparation thereof. <??>The new compounds of the general Formula I exhibit antitumor activity.

Description

The invention relates to a method for the preparation of M-nitroso-L- (bzta-chloroethyl) - carbamoyl peptides or their salts - new biologically active compounds that can be used in medicine.

The purpose of the invention is a method of obtaining new derivatives of N-nitrozo-N- - (beta-chloroethyl) carbamoyl peptides, which have a higher antitumor activity.

Chromatography was carried out in a thin layer of Kieselgel G (Merck) in the following solvent systems: A - chloroform: methanol 9: 1; B - n-butanol: ice on acetic acid: water 4: 1: 1; B-chloroform: methanol 8: 2} G - ethyl acetate: pyridine: ice on acetic acid: water and 60: 20: 6: 11, D - ethyl acetate: pyridine: formic acid: water 60: 20: 6-5,5; E - methanol: ethyl acetate 1: 3; W - ethyl acetate:

sc

  158

Hyiridine: ice on acetic acid:: | water 240: 20: 6: 11. : Example N-HHTpo30-N - (- - Chloroethyl) -carbamoyl-prolylvinnamide

one).

1.37 g (5 mmol) of prolyl-valine imide acetate are suspended in 5 ml of anhydrous dimethylformamide and 1.4 ml (10 mmol) of triethylamine are added with stirring and cooling of ij-OM. 1, 25 g (5 mmol) of N-nitro-ON - ((-chloroethyl) -carbamine acid-N-oxy-N hydroxy succinimide ester complex, the reaction mixture is stirred with 15 mi Ari 0 ° C, then diluted with ice water, the precipitated product from - :: (filtered, washed with cold soda on the filter. The product is dried in a desiccator over P. - s 0.91 g | (53%), mp. (decomposition), Rf t 0.8 ( B), 5 88 (M-nitrosogroup, 400 nm) ..

Calculated,%: C, 44.85; H 6.33; C1 10.19; N 20.13,

C, / Hij, (347.8). Found%: C, 83; H 6.35; |: i 9.86; N 19.76.,

i Example2. a) Alpha-N-nitrozo-K- (beta-chloroethyl) -carbamoyl-epsilon-tert.butyloxycarbonyl-lysyl-propyl in alin amy, g ,.

614 mg (1 mmol) epsilon-tert.bu-; toxicarbonyl-lysyl-prolylvaline-am.d 1-tosylate is dissolved in 10 ml of anhydrous

dimethylformamide, after which | 0.14 ml (1 mmol) of triethylamine, 274 m: (1 mmol) of K-nitroso-beta-chloro ethyl carbamoyl-p-nitrophenol and 136 ml (1 mmol) are added to the prepared solution 1-hydroxybenzotriazole. After 2 hours, dimethylformamide is distilled off in vacuo, the residue is dissolved in 30 ml of ethyl acetate and washed with water. The ethyl acetate layer is separated, dried, and evaporated. The residue is treated with anhydrous diethyl ether, the ether layer is separated by decantation, and the oil is dried.

The solid foam thus obtained is cleaned in a column filled with silica gel. The fractions were collected, and the trunk was distilled off in vacuo. The residue is ground to powder in the presence of anhydrous petroleum ether. A, filtered.

250 ml (43%) of compounds are obtained

Rf 0.90 (A), Rf 0.80 (B) 6 90 (N-nitrosogroup, 397 nm).

five

Q

0

five

0

five

0

55

Calculated,%: C1 6.15; N 17.02.

. (576,10).

Found,%: C1 6.00; N 16.61,

b) Alpha-) l-HHTpo3o-N- (beta-chloroethyl) -carbamoG-lysyl-prolyl-valine-amide hydrochloride (II).

200 mg of alpha-N-nitroso-L- (beta-chloroethyl) -carbamoyl-epsilon-tert. butoxycarbonyl-lysyl-prolyl-valine-amide is mixed with 6 ml 0.17 n. a solution of formic acid in hydrochloric acid and the solution is allowed to stand for 5 minutes, followed by evaporation. The residue is dissolved in water and extracted with ethyl acetate. The aqueous layer was separated and lyophilized. 140 mg of compound are obtained, Rf 0.10 (B).

Calculated,%: C 44.49; H 6.83; C 13.04; N 19.13.

, (512.44).

Found,%: C 44.51; H 6.80; C1 13.72; N 18.81.

PRI me R 3. Alpha epsilon-bis- - K-nitroso-M- (beta-chloroethyl) carbamoyl-J-lysyl-propyl-valine-amide (III).

460 mg (1 mmol) of lysyl-prolyl-α-vinamide diacetate are dissolved in 6 ml of dimethylformamide, then 0.28 ml (2 mmol) of triethylamine and 548 ml (2 mmol) of N-nitroso-beta-beta-chloro-ethane are added to the resulting solution - bamoyl-p-nitrophenol, and then 270 mg (2 mmol) of 1-hydroxybenzotriazole, after 2 h, dimethylformamide is distilled off in vacuum, the residue is dissolved in 30 ml of ethyl acetate, washed with water. The ethyl acetate layer is separated, dried, evaporated. The residue is treated with anhydrous diethyl ether, the ether is decanted, and the oil is dried. The resulting solid foam is cleaned in a column filled with silica gel. The fractions are collected and the solvent is distilled off in vacuo. The residue is ground to powder in the presence of anhydrous petroleum ether, filtered. 312 mg (51%) of product are obtained, Rf 0.80 (S), Rf 0.88, 5 185 (N-nitroso group, 397 nm).

Calculated,%: C 43.17; H 6.05; C1 11.59; N 20.61.

C iHjyNjOyCU (611,51).

Found,%: C 43.21; H 6.02; C1 11.18; N 20.07

Example 4. a) 0 - N-Nitrozo-N- / Ultrasonic chloroethyl) -carbamoyl-tertiary butyloxycarbonyl-tryptophyl-glycyl-lysyl-prolylvalinamide.

51586520

To a mixed solution of 1.2 g is evaporated in vacuo. The residue solution (1.74 mmol) of tertiary butyloxycarbo-ethyl acetate is washed with water.

The rot-tryptophenylglycyl-lysyl-prolyl-ethyl acetate layer is separated, dried

Valineamide and 10 ml of anhydrous dimethyl-with are evaporated. Obtain 1.29 g (80%)

,. /, J P QO. n Р z Q 1

frrmamid added at 0.24 ml

(1.74 mol) of triethylamine and 400 mg (1.6 mmol) of N-nitroso-M- (A-chloro-ethane 1) -carbamoyl-N-oxysuccinimide. After 0.5 h, the reaction mixture is evaporated, the residue is dissolved in ethyl acetate, and the solution is washed with water. The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated. Yield 1.2 g (85%), Rf (A).

Calculated,%: N 17,09; C1 4.33.

product. Rf 0.82 vB), 6 91 (N-nitrous group, 397 nm).

Calculated,%: C1 5.60; N 17.69.

C, H 5KzODS1

Found,%: C1 5.48; N 17.31.

b) Alpha-K-nitroso-L- (beta-chlorostil) -carbamoyl-glycyl-lysyl-prolyl-β-vinamide hydrochloride (V).

633 mg (1 mmol) of alpha-N-nitroes-5-H- (bzta-chloroethyl) -carbamoyl-epsilon-tert-butyloxycarbonyl-GLYCSh-1-LISZ7 5 u 1 (819.37).

Found,%: N 16.72; C1 4.01.

.6). Nitrozo T- (ft-chloroethyl) -carbamoyl-D-tryptophyl-glycyl-lysyl- -Prolyl-valyl-amidoacetate (IV).

819 mg (1 mmol) about-LN-nitroso-N- (y-chloroethyl) -carbamocl-tertiary. Butyloxycarbonyl-trypsfil-glycyl-α-lysyl-prolyl-valinamide is dissolved in 10 ml of 0.12 n. formic acid with hydrochloric acid, defend the solution for 5 minutes. then the solution is evaporated in vacuo. The residue is dissolved in water, extracted with ethyl L-acetate. The aqueous phase is separated, lyophilized, and the lyophilized is chromatographed on a column filled with silica gel, eluting with a mixture of ethyl acetate, pyridine, glacial acetic acid and water 60: 20: 6: 11. The eluate is evaporated. The residue is dissolved in water, lyophilized, 480 mg (63%) of the salts are obtained. Rf 0.25 (I); 90 (50 nm).

Calculated,%: C, 52.40; H 6.59; O 18.47; N 17.97, C1 4.55.

C, 4Hj, NioO, Cl (779.27).

Found,%: C 52.00; H 6.29; About 18.01; N 17.00; C1.

11Raymer5. a) Alpha-K-nitrozo-M- (beta-chloroethyl) -carbamoyl-epsilon-tert.butyloxycarbonyl glycyl-lysyl-prolyl-valinamide.

1.30 g (2J6 mmol) epsilrn-tert., Butyloxycarbonyl-glycyl-lysyl-prolyl-valinamide is dissolved in 13 ml of absolutized dimethylformamide,

Zyl-prolyl-valinamide is kept in a calm state for 5 minutes in 10 ml of 0.14 n. solution of formic acid 20 in hydrochloric acid, and then the solution is evaporated.

The residue is dissolved in water and extracted with ethyl acetate. The aqueous phase is lyophilized. 540 mg (95%) are obtained. 25 products, Rf 0.65 (D), g 84 (N-nitrosis group, 396 nm).

Calculated,%: C, 42.24; H 6.67; C1 12.45; N 19.67.

C -l iaNgOgClg (569.59). 30% found: C 42.22; H 6.69; C1 12.01; N 18.76.

PRI me R 6. N-Nitroso-M- (beta - chloroethyl) -carbamoyl-tryptophyl-norleucyl-alpha-asparagyl-phenylapanil-amide.

Under the conditions described in Example 3, 921 mg (1.5 mmol) tryptophyl-norlecyl-alpha-asparagyl-phenylalanilamide-Q hydrochloride, 0.21 ml O, 5 mmol) of triethylamine, 420 mg (1, 5 mmol) N-nitroso-beta-Ghlorztilkarbamoil - -p-nitrophenol and 200 mg (1.5 mmol) of 1-hydroxybenzotriazole. 800 mg ds of compound are obtained, yield 75%, Rf 0.8 (B),

Calculated,%: C 55.52; H 5.75; C1 is 4.97; N 15.71; N0 4.20. .OgCl (713.20). Found,%: C 55.50; H 5.77; 50 C1 4.58; N 15.32; N0 3.92.

Test A. The pharmacological activity of the proposed compounds is demonstrated by the results of a test.

35

  Research institutes using 650 mg (2.6 mmol) of 55 test compounds o6, /} - bis-Gk-nitM-nitroso-beta-chloroethylcarbamoyl-H-rozo-N- (| 5-chloroethyl) - carbamoyl-lysyl-oxysuccinimide and 0.36 ml (2.6 mmol) -prolyl-valinamide (on which further

triethylamine. After stirring in the text, the reaction mixture was boosted II) and o (-} -nitroso-1b ((3-chloro ", evaporated to the mixture for 15 min) for about 15 minutes). 1.29 g (80%

  P QO. n Р z Q 1

product. Rf 0.82 vB), 6 91 (N-nitrous group, 397 nm).

Calculated,%: C1 5.60; N 17.69.

C, H 5KzODS1

Found,%: C1 5.48; N 17.31.

b) Alpha-K-nitroso-L- (beta-chlorostil) -carbamoyl-glycyl-lysyl-prolyl-β-vinamide hydrochloride (V).

633 mg (1 mmol) of alpha-N-nitro-es- -H- (bzta-chloroethyl) -carbamoyl-epsilon- -t-butyloxycarbonyl-GLYCSh1-Lizil-prolyl-valinomide are kept in a calm state for 5 minutes in 10 ml of 0 , 14 n. solution of formic acid in hydrochloric acid, and then the solution is evaporated.

The residue is dissolved in water and extracted with ethyl acetate. The aqueous phase is lyophilized. 540 mg (95%) are obtained. product, Rf 0,65 (D), g 84 (N-nitroson group, 396 nm).

Calculated,%: C, 42.24; H 6.67; C1 12.45; N 19.67.

C -l iaNgOgClg (569.59). Found,%: C 42.22; H 6.69; C1 12.01; N 18.76.

EXAMPLE 6 S-Nitroso-M- (beta-chloroethyl) -carbamoyl-tryptophyl-nor-leucyl-alpha-asparagyl-phenylapanil-amide.

Under the conditions described in Example 3, 921 mg (1.5 mmol) tryptophyl-norlecyl-alpha-asparagyl-phenylalanilamide hydrochloride, 0.21 ml O, 5 mmol) triethylamine, 420 mg (1.5 mmol) N-nitroso-beta-Ghlorztilkarbamoil - -p-nitrophenol and 200 mg (1.5 mmol) of 1-hydroxybenztriazole. 800 mg of compound are obtained, yield 75%, Rf 0.8 (B),

Calculated,%: C 55.52; H 5.75; C1 is 4.97; N 15.71; N0 4.20. .OgCl (713.20). Found,%: C 55.50; H 5.77; C1 4.58; N 15.32; N0 3.92.

Test A. The pharmacological activity of the proposed compounds is demonstrated by the results of a test.

ethyl) -carbamoyl-lysyl-prolyl-valine-amnd hydrochloride (which is referred to hereinafter as compound III).

Table 1 shows the effect of compounds II and III on intraperitoneal implanted mice with leukemia. Compounds II and III turned out to be more r1 {scantium than 1, 3-bis - (/ 3-chloroethyl) -4 1-nitrosourea (hereinafter referred to as 1 BCNY) and (2-lorethyl) -3-1 itrouriedo -2 -2-deoxy-0-glucopyrano; ia (hereinafter referred to as chlorozoti-yin), used as a comparison of Whole substances (see tab.1), i Tumor graft material - | 0 ascites cells, intraperitoneally; conservative-female mice of the species (fDF; treatment - 3 days; toxicity - the fifth day of 4/6 survived; (|) Foam - MST-average period-in;

,, „/ MST processed group

Effect - T / C -:

MST control group

X 100; criterion - significant antitumor activity. ; Table 2 shows the pharmacological properties of compound III. I Pharmacological properties of compounds -) | 1eni III i Toxic dose of 133- too mg / kg intraperitoneally. The substance was used on mice infected with L-1210 (see table 2-7).

Table 3 shows the results of the study of the relationship between the effect and: the chemical structure of the compounds, Dialogue to the compound of formula (II), jfia in mice infected with L-1210 I leukemia. The results of the influence of the compounds of the formula (II) on the survival of mice infected with hay leukemia L-1210 are given in table 4.

B Table 5 shows the results of the effect of the compounds of formula (II) in combination with pharmaceutical agents on mice infected with leukemia L-1 2 IO

The above data indicates that the new peptide derivatives of formulas II and III are significantly more active and less toxic than BCNY.H chloro-tocin, used as control agents.

Test B. The effect of compound II on a solid tumor was investigated.

A) S180 tumor (subcutaneously).

A dosage of 100 or 5x20 mg / kg intraperitoneally (treatment started the day after transplantation) turned out to be ineffective.

B) Lewis pulmonary melanoma and B melanoma are tumors that give metastases in mice (i.m.)

The results are shown in table 6.

Test C.

Pharmacologists.

The effect of peptide-nitrosourea derivatives on the growth of experimental solid tumors and human tumor xenografts.

Objects:

a) experimental solid tumor of mouse S 180, pulmonary tumor of Lewis and melanoma of mouse B;

c) human tumor xenografts; melanol nonpigmented NT 18, colorectal adenocarcinoma HT 17, colorectal adenocarcinoma HT 22 (skin mucinosis), pancreatic carcinoma HT 27.

Experiments on experimental solid tumors: S al.

The tumor is cut into small pieces, and the pieces are transplanted under the skin of the back of the CLFP mice. Treatment begins the day after transplantation. Tumor size is measured with calipers and their volume is calculated from their diameter.

SV370 is administered intraperitoneally at doses of 100 or 5x20 mg / k (one dose for five days). No tumor growth was detected.

Lung's lung tumor (ALL).

This tumor is transplanted in the form of a suspension of cells in the femoral muscle of a C57B1 inbred mouse with 10 cells. Growing up. the tumor gives regular lung metastases. Treatment; begin the day after transplantation. The survival of the treated and control animals is observed.

Melanoma.

The tumor is transplanted under the skin of the back of the inbred C57B1 molecule in the form of tumor pieces. A growing tumor gives metastases in the lung. Treatment and evaluation are carried out in a similar manner as described in the Lewis lung tumor.

In accordance with the results, the connection of SV370 moderately lengthens the lifespan of animals including the cases of ALL and B melanoma. The order of efficacy in slowing tumor growth is basically equal to the effect of BCNY, used as a control compound (see table 7).

Human Xenograft Testing.

Pieces of serially transplanted tumors (matured ameteo, full irradiation 8.5 GY, restoration of the inert brain due to 5 «10 synthetic bone marrow cells) are transplanted under the skin of the back of an CBA / CA artificially depressed immune system. Treatment with an intraperitoneal method begins when tumors reach a diameter of 0.7-1.0 cm and their growth can be observed in progression. The diameters of the tumors are measured with calipers, and the volume is calculated based on their diameter (V c / b xD, D-average dia

meter). The order of efficacy of slowing tumor growth is determined on the basis of the difference in tumor volumes measured on day 21 after transplantation in the control and treated groups (each experimental group included at least 6 tumors), see Table. eight.

1 survival time

ILS% - - 5 BL; 5Э5 ™ 2 15251

 - time of entering the pin

Criterion: ILS.% - a significant effect of slowing tumor growth.

The results obtained show that the compounds obtained under the conditions of the proposed method are effective cytostatic agents, and more active than chloro-coticin and BCNY.

Claims (1)

Invention Formula The method of obtaining M-nitroso-M- (beta-chloroethyl) carbamoyl peptides or their salts of formula I N0 clCll..Nн, iU  U2 ;,.  -Pro-ValMl, -Lys-Pro-Vaim: Gly-Lys-Pro-ValNH,-Trp-Gly-Lys-Pro-ValllH,-Trp-Leu-Asp-PheNK, Where 1 R Experimental results show that peptide nitroso-urea derivatives significantly slow the growth of non-pigmented melioma, while their effect on the growth of colon and pancreas tumors is lower. The effect exerted by N-nitroso- (A-chloroethyl) -carbamoyl peptide fragments on the slowing down of tumor growth in L-1210 mice with leukemia is shown in Table 9. 15 Measurement of the effect of slowing tumor growth in L-1210 is carried out as follows: 10 L-1210 leukemine cells are transplanted intraperitoneally to DBA cells. Treatment begins the day after transplantation; The test compounds are added to a physiological solution of sodium chloride or to Tween 80 containing 2% buffer. 5% dimethyl sulfoxide. treated or their salts, differing from the fact that peptides of the general formula II are introduced into the interaction XHN-R, where X is hydrogen or boi; R is as defined, with an activated ester of the general formula III NU Cl — CH —ClI — N — CO — Q, D Q -n is nitrophenyl, halophenyl, oxysuccinimide or phthalimido-oxy group. moreover, when obtaining compounds 1-- where n, equimolar are used the amounts of the starting, if n 2, isodic II and III take in molar ratio of 1: 2, with the subsequent removal, if necessary, of protective groups and isolation of the target product in free form or in salt form. Table 1 sProValNfl -HCl-II  ) LysProValNlI-HCl Ticin s 40 30 150 100 65 40 200 130 70 40 48 32 16 8 Physiological NaCl solution 47.0 47.0 10.5 7.0 29.5 13.0 8.5 12.0 47.0 14.0 8.5 47.0 20.5 9.0 J / Both of you are sick. Note: Toxic dose of 133-200 mg / kg intraperitoneally. The substance is used on vehicles infected with .L-12 10. T a b l i.tsa 3 IV Q (NO) TrpGlyLysProValNH..2 V Q (NO) GlyLysProValNll ,,. KCl 783 783 175 783 492 217 142 200 783 233 142 783 342 150 -1.4 -0.8 -1.1 -0.7 -0.5 -0.7 -1.5 -0.6 -0.8 -0.3 -. , 3 -1,2 -0,8 + 1,2 +2,5 6/6 / 5 / 6/6 / 5 / 6/6 / 1 / 6/6 / 3 / 6/6 / 2 / 6/6 6/6, 6/6 / 2 / 6/6 / 3 / 6/6 6/6 5/6 / 3 / 6/6 6/6 10/10 table 2 ABOUT ten 50 Toxic 20 10 35 230 13 Intraperitoneally, use on the marked day after transplantation; survival of treated mice 7 -2 --- 9-25-21-йУ-2тСтНг survival time untreated X 100; Survival on the 60th day after transplantation. 325 325 1x25 2 44 1x50 2 44 1x50 2 o 1x10 2 77 77 44 1x50 3 33 1x20 3 70 1586520 Table 4 14 Table 5 70 60 15 1586520. 16 Table 6 Tumor Treatment Average survival time days eh eh Al TD eh eh Lewis 21.0 22.0 27.0 28.5 25.0 V melanoma 26.0 31.5 35.0 4 29 36 nineteen 21.5 35 42,061 34,031 Human tumor xenografts HT 18 (amelanotic melanoma) mean sign22, 5 50.0 - recounted 1.0 56,251,5 HT 22 (mucous colon carcinoma) 17 24,00,29 21,50,26 HT 1 7 (adenomatosis carcinoma of the colon) 16.5 14,750.0. 5.00.0 tagging Treatment time: upon reaching a tumor volume of about 8 Treatment method: intraperitoneally; TD doubling the tumor volume; TD: TDCO  td; g Ke - processed; Co - control, Diabetes growth retardation ILS,% 4 29 36 nineteen 21.5 35 Control 21, P Compound III ILS is the duration of survival time, The effect of slowing tumor growth,%, with the introduction of a substance in the amount, mg / kg Efficiency slowing down the growth of 100 - - S5 - eEp t opt for 2J day; about o6p 0 days the tumor volume of the control tumor on day 21 volume con. About days. (IP.Q. means complete tumor growth retardation). Table 26.0 Table 8 Q (NO) - C1-C ..- N (NO) -COT tables